Desformylflustrabromine (dFBr; 1) and desformylflustrabromine-B (dFBr-B; 2) have been previously isolated from natural sources, and the former has been demonstrated to be a novel and selective positive allosteric modulator of alpha4beta2 nicotinic acetylcholine (nACh) receptors. The present study describes the synthesis of water-soluble salts of 1 and 2, and confirms and further investigates the actions of 1 and 2 using two-electrode voltage clamp recordings.

The nicotinic acetylcholine receptor (AChR) and the serotonin type 3 receptor (5HT 3 R) are members of the ligand-gated ion channel gene family. Both receptors are inhibited by nanomolar concentrations of d-tubocurarine (curare) in a competitive fashion. Chemical labeling studies on the AChR have identified tryptophan residues on the ␥ (␥Trp-55) and ␦ (␦Trp-57) subunits that interact with curare. Comparison of the sequences of these two subunits with the 5HT 3 R shows that a tryptophan residue is found in the homologous position in the 5HT 3 R (Trp-89), suggesting that this residue may be involved in curare-5HT 3 R interactions. Site-directed mutagenesis at position Trp-89 markedly reduces the affinity of the 5HT 3 R for the antagonists curare and granisetron but has little effect on the affinity for the agonist serotonin. To further examine the role of this region of the receptor in ligand-receptor interactions, alanine-scanning mutagenesis analysis of the region centered on Trp-89 (Thr-85 to Trp-94) was carried out, and the ligand binding properties of the mutant receptors were determined. Within this region of the receptor, curare affinity is reduced by substitution only at Trp-89, whereas serotonin affinity is reduced only by substitution at Arg-91. On the other hand, granisetron affinity is reduced by substitutions at Trp-89, Arg-91, and Tyr-93. This differential effect of substitutions on ligand affinity suggests that different ligands may have different points of interaction within the ligand-binding pocket. In addition, the every-other-residue periodicity of the effects on granisetron affinity strongly suggests that this region of the ligand-binding site of the 5HT 3 R (and by inference, other members of the ligand-gated ion channel family) is in a ␤-strand conformation.The serotonin type 3 receptor (5HT 3 R) 1 is a member of a superfamily of ligand-gated ion channels, which includes the muscle and neuronal nicotinic acetylcholine receptor (AChR), the glycine receptor, and the ␥-aminobutyric acid type A receptor (1-3). Like the other members of the gene superfamily, the 5HT 3 R exhibits a large degree of sequence similarity, and thus presumably structural homology, with the AChR (4). Chimeras containing the amino-terminal domain of the ␣7 neuronal AChR and the carboxyl-terminal domain of the 5HT 3 R form functional ligand-gated channels with ligand specificities characteristic of AChR and permeability properties of 5HT 3 Rs (5). These experiments suggest that the AChR and 5HT 3 R have quite similar structures and signal transduction mechanisms.A large number of studies have been conducted to elucidate the structure of the ligand-binding site for AChRs (6, 7); however, very little is known about the interactions between ligands and the 5HT 3 R. A common competitive antagonist of the AChR and 5HT 3 R is d-tubocurarine (curare), which inhibits both receptors at nanomolar concentrations (8, 9). If both receptors are similar in structure, it seems reasonable to assume that similar regions of the receptors may...

Neuronal nicotinic acetylcholine receptors (nAChRs) are members of the Cys-loop superfamily of ligand-gated ion channels. nAChRs are involved in modulating nicotinic-based signal transmission in the central nervous system and are implicated in a range of disorders. Desformylflustrabromine (dFBr) is a positive allosteric modulator that potentiates ␣4␤2 nAChRs. It has been reported that dFBr is selective for the ␣4␤2 receptor relative to other common nAChR subtypes (Neurosci Lett 373: 144 -149, 2005). Coapplication of dFBr with acetylcholine (ACh) produces a bell-shaped dose-response curve with a peak potentiation of more than 265% (Bioorg Med Chem Lett 17:4855-4860, 2007) at dFBr concentrations Ͻ10 M and inhibition of responses at concentrations Ͼ10 M. The potentiation and inhibition components of dFBr-modulated responses were examined by using two-electrode voltage clamp and human ␣4␤2 nAChRs expressed in Xenopus laevis oocytes. Currents to both partial and full agonists were potentiated by dFBr. Responses to low-efficacy agonists were potentiated significantly more than responses to high-efficacy agonists. Antagonist pIC 50 values were unaffected by coapplication of dFBr. In addition to its potentiating effects, dFBr was able to induce current spikes when applied to desensitized receptors, suggestive of a shift in equilibrium from the desensitized to open conformation. In contrast to potentiation, inhibition of ACh responses by dFBr depends on membrane potential and is probably the result of open-channel block by dFBr and ACh. Our data indicate distinct mechanisms for the potentiation and inhibition components of dFBr action. dFBr could prove useful for therapeutic enhancement of responses at ␣4␤2-containing synapses.

Nicotinic α4β2 receptors are the most abundant subtypes of nicotinic acetylcholine receptors (nAChRs) expressed in brain regions implicated in obsessive compulsive disorder (OCD). These receptors are known to modify normal and addictive behaviors by modulating neuronal excitability. Desformylflustrabromine (dFBr) is a novel, positive allosteric modulator (PAM) of high acetylcholine sensitivity (HS) and low acetylcholine sensitivity (LS) α4β2 nAChRs. The present study tested the hypothesis that positive allosteric modulation of α4β2 receptors by dFBr will attenuate compulsive-like behavior in a non-induced compulsive-like mouse model. Male mice (Mus musculus) selected for compulsive-like nesting behavior (NB; 48 animals; 12 per group) received acute (once) and chronic (every day for 32 days) subcutaneous injection of dFBr at 2, 4 and 6 mg/kg doses. Saline was used as a control (0 mg/kg). Compulsive-like NB was assessed after 1, 2, 3, 4, 5 and 24 h, while compulsive-like marble burying (MB) and anxiety-like open field (OF) behaviors were performed 2 h after dFBr administration. In the acute administration protocol, dFBr dose dependently attenuated NB and MB. Rapid effects (1–2 h after drug administration) of dFBr on MB and NB were observed for the chronic administration which was in congruence with the acute study. Chronic administration also revealed sustained suppression of NB by dFBr following 5 weeks of treatment. In both the acute and chronic regimen dFBr did not modulate OF behaviors. This research demonstrates the novel role of positive allosteric modulation of α4β2 nicotinic receptors by dFBr as a translational potential for OCD.

Rabies virus induces drastic behaviour modifications in infected hosts. The mechanisms used to achieve these changes in the host are not known. The main finding of this study is that a region in the rabies virus glycoprotein, with homologies to snake toxins, has the ability to alter behaviour in animals through inhibition of nicotinic acetylcholine receptors present in the central nervous system. This finding provides a novel aspect to virus receptor interaction and host manipulation by pathogens in general. The neurotoxin-like region of the rabies virus glycoprotein inhibited acetylcholine responses of α4β2 nicotinic receptors in vitro, as did full length ectodomain of the rabies virus glycoprotein. The same peptides significantly altered a nicotinic receptor induced behaviour in C. elegans and increased locomotor activity levels when injected into the central nervous system of mice. These results provide a mechanistic explanation for the behavioural changes in hosts infected by rabies virus.